Pseudomonas sp. and application thereof in degrading octyl p-methoxycinnamate

Abstract

The application discloses an octyl p-methoxycinnamate degrading bacterium, Pseudomonas sp. Pseudomonas OMC5, the preservation information of which is as follows: a preservation unit is Guangdong Microbial Culture Collection Center (GDMCC), a preservation date is November 25, 2025, a preservation address is the fifth floor of No. 59 building, Guangdong Microorganism Institute, 100, Middle Jianli Road, Guangzhou, a postal code is 510070, and a preservation number is GDMCC NO. 67353. The application separates Pseudomonas sp. Pseudomonas OMC5 with good degradation effect on octyl p-methoxycinnamate from sludge of a Guangzhou sewage treatment plant, and the Pseudomonas sp. OMC5 can be used for degrading and removing octyl p-methoxycinnamate. An economic and green method is provided for removing the new type of organic pollutants.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment, specifically relating to a strain of octyl methoxycinnamate degrading bacteria. Background Technology

[0002] Octyl p-methoxycinnamate is a commonly used organic ultraviolet absorber. It is widely added to various personal care products, such as cosmetics and sunscreens, to protect skin from sunlight, as well as other consumer products such as plastics, textiles, food packaging, tires, and detergents to prevent photodegradation. It is frequently detected in the environment due to recreational activities such as swimming or insufficient removal capacity in wastewater treatment plants. Due to its high lipophilicity (logKow > 4), octyl p-methoxycinnamate entering the aquatic environment readily accumulates in organisms and has biomagnification potential. Long-term accumulation of octyl p-methoxycinnamate can interfere with the endocrine system of organisms, seriously threatening their development and reproduction. For example, octyl p-methoxycinnamate can reduce thyroid hormones in adult male and larval zebrafish, and may also affect the transcription of neurotoxicity-related genes in larval fish and nephrotoxicity-related genes in adult male fish (Seoyoon Chu, Ba Reum Kwon, Young-Min Lee, Kyung-DukZoh, Kyungho Choi, Effects of 2-ethylhexyl-4-methoxycinnamate (EHMC) on thyroid hormones and genes associated with thyroid, neurotoxic, and nephrotoxic responses in adult and larval zebrafish (Danio rerio), Chemosphere, Volume 263, 2021, 128176).

[0003] Currently, research on the removal of octyl methoxycinnamate is limited. Although wastewater treatment plants have some removal effect on octyl methoxycinnamate, the frequent detection of organic ultraviolet absorbers such as octyl methoxycinnamate in the environment indicates a lack of targeted methods for removing these emerging organic pollutants. Considering both environmental and economic factors, microbial degradation is considered an economical and green method for removing organic matter from the environment. Domestic and international research on the degradation of octyl methoxycinnamate by bacterial strains is limited, and further research is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of octyl methoxycinnamate degrading bacteria.

[0005] The octyl methoxycinnamate degrading bacteria of the present invention are Pseudomonas ( Pseudomonas sp.)OMC5, its deposit information: depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: November 25, 2025, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postcode: 510070, deposit number: GDMCC NO.67353.

[0006] The present invention also provides a microbial inoculant containing the above-mentioned Pseudomonas OMC5 or its fermentation broth as an active ingredient.

[0007] Preferably, the microbial agent can be a microbial additive.

[0008] The present invention also provides the application of the above-mentioned Pseudomonas OMC5 or microbial agents in the degradation of octyl p-methoxycinnamate.

[0009] Preferably, the p-methoxycinnamate octyl ester includes p-methoxycinnamate octyl ester in various environments, such as soil and water environments.

[0010] The present invention also provides a method for degrading octyl p-methoxycinnamate, which involves inoculating the above-mentioned Pseudomonas OMC5 or microbial agent into an environment containing octyl p-methoxycinnamate, and using Pseudomonas OMC5 to degrade octyl p-methoxycinnamate.

[0011] This invention isolates a strain of Pseudomonas aeruginosa that exhibits good degradation effects on octyl p-methoxycinnamate from sludge of a wastewater treatment plant in Guangzhou. Pseudomonas OMC5 (sp.) can be used to degrade and remove octyl methoxycinnamate, providing an economical and green method for removing this type of novel organic pollutant.

[0012] Pseudomonas sp. OMC5, its deposit information: depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: November 25, 2025, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postcode: 510070, deposit number: GDMCC NO.67353. Attached Figure Description

[0013] Figure 1 This is a colony morphology diagram of Pseudomonas OMC5;

[0014] Figure 2 This is a graph showing the degradation effect of Pseudomonas OMC5 on octyl methoxycinnamate. Detailed Implementation

[0015] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0016] Example 1: Domestication, isolation and identification of octyl methoxycinnamate-degrading bacteria OMC

[0017] Bacterial sources were collected from sludge at a wastewater treatment plant in Guangzhou. 30 mL of sludge was inoculated into a 250 mL Erlenmeyer flask containing 120 mL of modified MSM liquid medium. The modified MSM liquid medium (plates contain 15 g / L agar) composition (1 L): 1 L mini-Q water, 9 g K₂HPO₄·3H₂O, 3 g KH₂PO₄, 0.5 g NaCl, 1 g NH₄Cl, 0.25 g MgSO₄·7H₂O, 0.014 g anhydrous CaCl₂, 1 mL trace element solution (0.636 g / L NaHCO₃, 0.1 g / L ZnSO₄·7H₂O, 0.05 g / L CuSO₄·5H₂O, 0.01 g / L (NH₄)₆Mo₇O₇). 24 ·4H2O, 0.25 g / L CoCl2·6H2O, 0.25 g / L FeSO4·7H2O, 0.15 g / L MnCl2·4H2O, 1 mL vitamin stock solution (20 mg / L biotin, 20 mg / L floic acid, 100 mg / L pyridoxine hydrochloride, 50 mg / L riboflavin, 50 mg / L thiamine, 50 mg / L nicotinic acid, 50 mg / L pantothenic acid, 1 mg / L vitamin B12, 50 mg / L p-aminobenzoic acid, 50 mg / L thioctic acid). Octyl methoxycinnamate standard was added to bring the concentration of octyl methoxycinnamate in the wastewater to 1 mg / L. After sealing with sealing film, the mixture was acclimatized and cultured at 25°C in the dark. When the removal rate of octyl methoxycinnamate in the system was above 80%, a higher concentration of octyl methoxycinnamate was added for the next acclimatization. The acclimatization concentration was gradually increased from 1 mg / L, 2 mg / L, 4 mg / L, 5 mg / L, and 10 mg / L.

[0018] After acclimatization was stopped, the acclimatization solution was serially diluted and spread onto LB and modified MSM plates for further cultivation. Bacteria with different morphologies, colors, and odors were streaked as much as possible. After multiple streaks, single bacteria were screened out and added to modified MSM liquid medium containing only octyl methoxycinnamate for cultivation. Based on the residual amount of octyl methoxycinnamate in the medium, single bacteria with the ability to degrade octyl methoxycinnamate were screened. The bacterial strain was preserved using the glycerol method (1000 μL of bacterial culture was placed in a 2 mL cryovial, 500 μL of sterilized 60% glycerol solution was added, vortexed, and stored at -80℃). Samples were sent for sequencing as soon as possible to identify the bacterial strain, obtaining strain OMC5.

[0019] The colonies of strain OMC5 are round, smooth, pale yellow, moist, and sticky. Figure 1 The biological characteristics are: oxidase positive, catalase positive, Gram-negative, oxidative metabolism, obligate aerobic, and this bacterium can degrade octyl p-methoxycinnamate under aerobic conditions.

[0020] The OMC5 strain was sent to Beijing Liuhe BGI Genomics Co., Ltd. for 16S rDNA identification. Sequence amplification was performed using forward primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (1500bp, SEQ ID NO: 2) and reverse primer (1492R): 5'-TACGGCTACCTTGTTACGACTT-3' (1500bp, SEQ ID NO: 3). Using total bacterial DNA as a template, the PCR reaction program was as follows: 96℃ pre-denaturation for 5 min; 96℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; final extension at 72℃ for 5 min, and storage at 4℃. The obtained 16S rDNA sequence (as shown in SEQ ID NO: 1) was entered into GenBank for BLAST homology comparison. The comparison revealed that strain OMC5 (the strain that degraded octyl methoxycinnamate the fastest in this experiment) was similar to... Pseudomonas chengduensis strain MBR showed the highest homology, reaching 100%. Based on morphological, physiological and biochemical indicators, and 16S rDNA gene sequence analysis, strain OMC5 was preliminarily identified as... Pseudomonas sp., named Pseudomonas ( Pseudomonas sp.)OMC5, its deposit information: depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: November 25, 2025, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, Postcode: 510070, deposit number: GDMCC NO.67353.

[0021] Example 2: Degradation effect of Pseudomonas OMC5 on octyl p-methoxycinnamate in MSM liquid medium

[0022] Prepare modified MSM liquid medium, sterilize and cool to room temperature, and aseptically dispense into 500mL Erlenmeyer flasks. Add octyl methoxycinnamate to achieve initial concentrations of 0.5, 1, 5, and 10 mg / L. The experiment is divided into two groups: (1) Infected group: Pseudomonas OMC5 is inoculated into modified MSM liquid medium and OD is set. 600 =0.05, (2) Aseptic control group, without inoculation with Pseudomonas OMC5. All conical flasks were sealed with sealing film, protected from light, and placed in an incubator at 25℃; three replicates were set up for both experimental and control groups, and the residual amount of p-methoxycinnamate in the culture medium was continuously measured to analyze the degradation ability of Pseudomonas OMC5 on p-methoxycinnamate. The results are as follows. Figure 2 As shown in the figure, *Pseudomonas* OMC5 removed over 80% of 0.5 mg / L octyl methoxycinnamate within 24 hours, over 95% of 1 mg / L octyl methoxycinnamate within 48 hours, over 95% of 5 mg / L octyl methoxycinnamate within 7 days, and over 95% of 10 mg / L octyl methoxycinnamate within 12 days. In the control group without *Pseudomonas* OMC5, there was no significant change in the concentration of octyl methoxycinnamate.

[0023] This case demonstrates that Pseudomonas OMC5 exhibits good degradation effects on both low and high concentrations of octyl p-methoxycinnamate in modified MSM liquid medium.

[0024] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, combinations, simplifications, or adjustments made under the spirit and core principles of the present invention should be considered equivalent technical solutions and are all included within the scope of protection of the present invention.

Claims

1. Pseudomonas ( Pseudomonas sp.)OMC5, accession number: GDMCC NO.67353.

2. A microbial inoculant, characterized in that, It contains the Pseudomonas OMC5 of claim 1 or its fermentation broth as an active ingredient.

3. The use of the Pseudomonas OMC5 of claim 1 or the microbial agent of claim 2 in the degradation of octyl p-methoxycinnamate.

4. The application according to claim 3, characterized in that, The p-methoxycinnamate octyl ester mentioned is p-methoxycinnamate octyl ester in soil or water environment.

5. A method for degrading octyl p-methoxycinnamate, characterized in that, The method involves inoculating the Pseudomonas OMC5 of claim 1 or the microbial agent of claim 2 into an environment containing octyl p-methoxycinnamate, and using Pseudomonas OMC5 to degrade octyl p-methoxycinnamate.

6. The method according to claim 5, characterized in that, The p-methoxycinnamate octyl ester mentioned is p-methoxycinnamate octyl ester in soil or water environment.

Images